Changes in levels of bio-chemicals and secondary
metabolites during peak stress in field cassava
Ephraim Nuwamanya, Patrick R Rubaihayo, Settumba
Mukasa, Samuel Kyamanywa, Robert Kawuki,
Joseph Hawumba and Yona Baguma*
NaCRRI/MAK
First Bio-Innovate Regional Scientific Conference
United Nations Conference Centre (UNCC-ECA)
Addis Ababa, Ethiopia, 25-27 February 2013

Cassava with drought tolerance
The rationale for breeding cassava with drought
tolerance is built in the fact that there is need to
improve the inherent capacity of cassava
as a food security and famine crop
particularly in Sub-Saharan Africa, where millions of
people suffer from chronic food shortages
(Elsharkway, 1993)

Drought and temperature stress
• Drought stress =
–lack of precipitation over a period
of time (Willwhite et al., 2007) or severe
low soil moisture that denies plant
access to water
• Heat stress = increase in Temp above
threshold for a period sufficient to
cause damage to plant
• Mitigation measures include
avoidance or tolerance mechanisms

Wang et al., , Planta, 2003

Problem statement
• In Uganda, no cassava varieties have been
selected for tolerance to heat &moisture stress
• No efforts have been put forward to understand
mechanisms behind observed
tolerance/resistance
• This has hampered breeding efforts to improve
cassava for tolerance to a number of abiotic
stresses

Materials and experimental Layout
• 20 Cassava varieties selected from different parts
of country (www.selectionsfordrought tolerance
study.ne)
• Field experiment will be set up in Kasese Western
Uganda (Gmap Kasese)
• Lay out will be RCBD with 4 replications, 2 stressed
and 2 control (irrigated) (exptal layout)
•

Why Kasese?. Weather Characteristics
In addition to receiving low rainfall, the trial site is in the rift valley experiencing higher
than average temperatures compared to the rest of Kasese district and the shallow soils
allow for easy drainage of water increasing moisture stress

Soil properties at the trial site
Property Units Content Range
pH - 6.53 6.4-6.7
Organic matter % 4.01 3.5-4.8
Nitrogen % 0.22 0.2-0.25
Phosphorus Ppm 23.80 6.0-55
Calcium Ppm 4090.26 2316.4-7573
Magnesium Ppm 882.3 346.3-1467.5
Potassium Ppm 383.98 194.8-675.1
Boron Ppm 1750 12.87-26.8
Zinc Ppm 0.58 0.2-1.1
Copper Ppm 10.23 8.2-13.8
Manganese Ppm 156.34 82.0-235.7
Iron Ppm 457.57 380.7-1786
Sand % 53.47 49.8-59.8
Clay % 21.90 24.9-30.9
Silt % 18.63 15.3-21.3

Tissue and Organ Sampling Guide
• Leaves
• All measurements to be done on 5th fully
expanded leaf unless otherwise
– Leaf discs will be cut out of the 5th fully
expanded leaf
– Preservation in liquid nitrogen during transfer
to the lab.
• Stems and roots
• All measurements to be whole plant parts
sampled by taking samples on the distal, middle
and posterior parts of the plant parts
– Discs will be cut out of the root
– Preservation in liquid nitrogen

Carbohydrates, Proteins and Pigments
• Soluble sugars (hexoses) (Dubois et al.,
1956; modified)
• Starch content (Megazyme starch kit)
• Cyanide content according to Onwuka
– Cyanide (mg/g) = Absorbance x GF x DF
Sample weight
– Where: GF = gradient factor and DF =
dilution factor
• Concentrations of chlorophylls and carotenoids
• determined in 100 % acetone (Wettshtein
1957)
• at 662, 644 and 440.5 nm for chlorophyll a,
chlorophyll b and carotenoids, respectively.

Establishment of peak stress
• Described as the time between the onset of
severe drought up to when relative humidity
levels go up.
• A period of less than 40% RH, less than 20%
soil moisture and daily average temperatures
above 340C.
• A period between in 9MAP and 11MAP
spanning over a five week period

Plate 2: Physiological differences in the stay green (A), Susceptible (B) and early recovering (C) varieties pre-stress (1), 3 weeks post stress (2), 5
weeks post stress (3) and 7weeks post stress (4)
A1
C1
B1
A4
A2
A3
B2 B3 B4
C3
C2 C4

Plate 1: Contrasting phenotypes dependent on physiological
mechanism for tolerance to drought. A: Stay green, B: Susceptible and
C: Early recovering varieties at 6-8 weeks post stress (peak stress)
A B C

Phenotypic observations during peak stress

Changes in starch content and FRS
Group H1SC H1FRS H2SC H2FRS H3SC H3FRS H4SC H4FRS RSC RFRS R2SC R2FRS
All Varieties 0.125 ± 0.007 0.151± 0.027 0.137± 0.02 0.217± 0.028 0.070± 0.029 0.179± 0.026 0.087± 0.013 0.206± 0.101 -0.017 0.013 0.6 0.308
Stay Green 0.133± 0.005 0.134± 0.029 0.177± 0.039 0.240± 0.008 0.075± 0.015 0.182± 0.012 0.077± 0.005 0.231± 0.027 -0.023 0.023 0.74 0.377
Early Recovering 0.106± 0.026 0.171± 0.033 0.111± 0.006 0.195± 0.004 0.071± 0.000 0.166± 0.023 0.075± 0.002 0.142± 0.001 -0.014 -0.012 0.64 0.48
Susceptible 0.106± 0.016 0.168± 0.029 0.134± 0.002 0.227± 0.003 0.070 ± 0.013 0.170± 0.069 0.071± 0.010 0.239± 0.004 -0.017 0.018 0.502 0.29
Table 1: H=Harvest 1, 2,3,and 4 corresponding to week 2, 4, 6 and 8 during stress. SC=Starch Content, FRS=Free Reducing Sugars, R=Rate of change in SC or FRS during the 8 weeks of stress.
R2 = Regression Coefficient for changes in the different parameters during the stress period.
• Differences in rate of reduction or accumulation
of starch and Reducing sugars were observed
• Rate of reduction in starch content high for SGV but low for ERV
• Rate of increase in reducing sugars was high for SGV and SV but low
(infact –ve gradient) for ERV-low amylase activity for ERV
• No remobilisation from the root observed for ERV compared to SGV
• Differences in mechanism of tolerance confirmed

y = 0.02x + 0.3573
R² = 0.4775
0
0.1
0.2
0.3
0.4
0.5 A
Reducing Sugars
Linear (Reducing
Sugars)
y = -0.0718x + 0.6025
R² = 0.1433
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9 B
Reducing Sugars
Linear
(Reducing
Sugars)
y = 0.1104x + 0.2565
R² = 0.9271
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
C
Reducing Sugars
Linear (Reducing
Sugars)
y = -0.005x + 0.4075
R² = 0.0163
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5 D
reducing
Sugars
Linear
(reducing
Sugars)
A=Changes in Reducing Sugars among stay green varieties B=Changes in Reducing Sugars for Early recovering varieties
C=Changes in Reducing Sugars for susceptible variety. D=Changes in reducing Sugars for all the varieties
Results: Free Reducing Sugars (RS)

Changes in peel and fresh root cyanide contents
Variety group H1CR H1CP H2CR H2CP H3CR H3CP H4CR H4CP H5CR H5CP RCR RCP R2CR R2CP
All varieties 0.332± 0.171 0.522±
0.207
0.339± 0.175 0.552±0.4
51
0.639±
0.513
1.269±0.
781
0.349±
0.158
0.557± 0.329 0.258±0.201 0.646± 0.613 -0.014 0.025 0.023 0.016
Stay green varieties 0.393± 0.201 0.393±
0.132
0.309± 0.029 0.298±
0.144
0.549±
0.202
0.984±
0.448
0.347±
0.125
0.501± 0.344 0.356± 0.327 0.646± 0.571 -0.003 0.071 0.003 0.176
Early recovering
varieties
0.301± 0.022 0.602±
0.216
0.245± 0.087 0.535±
0.072
0.642±
0.034
1.457±
0.327
0.246±
0.237
0.407± 0.196 0.087± 0.010 0.149± 0.014 -0.043 -0.103 0.108 0.110
Susceptible
varieties
0.168± 0.134 0.386±
0.121
0.381± 0.022 0.510±
0.038
0.405±
0.027
1.605±
0.022
0.301±
0.104
0.667± 0.137 0.070± 0.118 0.286± 0.087 -0.028 -0.004 0.093 0.002
Table 2: Changes in the cyanide content levels from onset of peak stress up to recovery. H=harvest, CP=Cyanide content for the peel; CR=Cyanide content for the root. R= rate of change;
R2=Regression
• Although differences in rate of increase in cyanide were
observed, peel cyanide content was the most important in
making inferences on plants physiological behavior
• While increments were observed for peel cyanide content in
SGV, reductions were observed in ERV
• Minor reductions were observed for fresh root cyanide content
in SGV over the peak stress period while significant reductions
were observed for ERV
• No significant differences were observed for cyanide in SV

Changes in Cyanide Content
y = 0.0253x + 0.6333
R² = 0.016
y = -0.0138x + 0.4232
R² = 0.023
0
0.2
0.4
0.6
0.8
1
1.2
1.4
MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9)
A
CnP Peel
CnP Root
Linear (CnP Peel)
Linear (CnP Root)
y = 0.0713x + 0.3497
R² = 0.1758
y = -0.0031x + 0.3993
R² = 0.0027
0
0.2
0.4
0.6
0.8
1
1.2
MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9)
B
CnP Peel
CnP Root
Linear (CnP Peel)
Linear (CnP Root)
y = -0.1034x + 0.9402
R² = 0.1097
y = -0.0427x + 0.4323
R² = 0.1084
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9)
C
CnP Peel
CnP Root
Linear (CnP Peel)
Linear (CnP Root)
y = -0.0043x + 0.7037
R² = 0.0002
y = -0.0276x + 0.3478
R² = 0.0931
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9)
D
CnP Peel
Cnp Root
Linear (CnP Peel)
Linear (Cnp Root)
A=Cyanide changes for all varieties throughout the stress period, B=Cyanide Change for stay green varieties, C= Cyanide change for early recovering
varieties, D=Cyanide change for susceptible variety

Total Pigments; Chla, Chlb, Cart
y = 0.0211x + 0.151
R² = 0.7326
y = 0.0052x + 0.079
R² = 0.3004
y = -0.0204x + 0.464
R² = 0.3245
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Harvest1 Harvest 2 Harvest 3 Harvest 4
Total pigment content
Chla
Chlb
cart
Linear (Chla)
Linear (Chlb)
Linear (cart)
y = 0.0136x + 0.1765
R² = 0.5475
y = 0.0041x + 0.0804
R² = 0.1281
y = -0.0405x + 0.5145
R² = 0.6803
0
0.1
0.2
0.3
0.4
0.5
0.6
Harvest1 Harvest2 Harvest 3 Harvest 4
Pigments for stay greens
Chla
Chlb
cart
Linear (Chla)
Linear (Chlb)
Linear (cart)
y = 0.0456x + 0.072
R² = 0.9779
y = 0.0153x + 0.042
R² = 0.9619
y = 0.0254x + 0.313
R² = 0.8683
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Harvest1 Harvest2 Harvest 3 Harvest 4
Graph for early recovering genotypes
Chla
Chlb
cart
Linear (Chla)
Linear (Chlb)
Linear (cart)
y = 0.0073x + 0.169
R² = 0.0298
y = 0.0004x + 0.084
R² = 0.0004
y = -0.0141x + 0.446
R² = 0.0455
0
0.1
0.2
0.3
0.4
0.5
0.6
Harvest1 Harvest2 Harvest 3 Harvest 4
Pigments for susceptible
Chla
Chlb
cart
Linear (Chla)
Linear (Chlb)
Linear (cart)

Bound Reducing Sugars/Total carbohydrate
• There was an increase in total metabolisable
carbohydrates
• Storage/structural carbohydrates reduced with
progressive stress
• Among SGV and SV, high increments in metabolisable
carbohydrates were coupled to high reductions in
storage carbohydrates
• Among ERV, reductions were observed in free
metabolisable carbohydrates while slight reductions
were observed for storage carbohydrates

Changes in carbohydrate Profiles
y = 0.0127x + 0.1565
R² = 0.3084
y = 0.039x + 0.174
R² = 0.4326
y = -0.0174x + 0.15
R² = 0.6
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Harvest 1 Harvest 2 Harvest 3 Harvest 4
Total carbohydrate metabolite changes
Free RS
Bound RS
Starch Content
Linear (Free RS)
Linear (Bound RS)
Linear (Starch Content)
y = 0.0233x + 0.1385
R² = 0.3771
y = 0.0094x + 0.251
R² = 0.015
y = -0.0232x + 0.164
R² = 0.7434
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Harvest 1 Harvest 2 Harvest 3 Harvest 4
Carbohydrate profiles for Stay green
Free RS
Bound RS
Starch Content
Linear (Free RS)
Linear (Bound RS)
Linear (Starch Content)
Linear (Starch Content)
y = -0.0116x + 0.1975
R² = 0.4748
y = -0.006x + 0.216
R² = 0.0676
y = -0.0138x + 0.1265
R² = 0.634
0
0.05
0.1
0.15
0.2
0.25
0.3
Harvest 1 Harvest 2 Harvest 3 Harvest 4
Carbohydrate profile for Early recovering
Free RS
Bound RS
Starch Content
Linear (Free RS)
Linear (Bound RS)
Linear (Starch Content)
y = 0.0156x + 0.162
R² = 0.2918
y = -0.0054x + 0.278
R² = 0.0026
y = -0.0169x + 0.1375
R² = 0.5023
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Harvest 1 Harvest 2 Harvest 3 Harvest 4
Carbohydrate profiles for susceptible varieties
Free RS
Bound RS
Starch Content
Linear (Free RS)
Linear (Bound RS)
Linear (Starch Content)
Linear (Starch Content)

Conclusions
• Peak stress is characterized by varied
physiological responses involving modification in
carbohydrate and Nitrogen metabolism
• Varieties respond differently during peak stress
showing varied mechanisms of tolerance to stress
• Important parameters for consideration in
understanding the levels of tolerance include
total bound and free sugars, cyanide content as a
secondary metabolite, and free metabolisable
sugars

•Thank you
•Asante sana